Mitogenic Activity of Cytoplasmic Membranes Isolated from L-Forms of Staphylococcus aureus

Cytoplasmic membranes of L-forms of Staphylococcus aureus exerted a strong mitogenic effect on splenocytes of athymic nude mice as well as normal mice, while a cytoplasmic fraction of the same bacteria did not show definite mitogenicity. The mitogenic principle(s) of the membrane fraction was resistant to treatment with trypsin and was heat stable (at 100 C for 10 min). The active principle(s) in the insoluble residue of the membrane fraction digested with trypsin was not extracted with cold acetone, but could be solubilized by extraction with a cold chloroform-methanol mixture (2:1, v/v). The mitogenic principle(s) in the extract was fractionated by silicic acid column chromatography. Among five fractions separated by chromatography, fractions eluted with chloroform-methanol mixtures (1:1 and 1:20, v/v) were found to be strongly mitogenic. The cytoplasmic membranes of the L-forms also exerted a definite mitogenic effect on guinea pig splenocytes, but not on the thymocytes.     

Biosynthesis, processing and half-life of P-glycoprotein in a human multidrug-resistant KB cell

The biosynthesis, processing, and half-life of the drug efflux pump, P-glycoprotein, were studied in human multidrug-resistant KB (KB-C2) cells selected for resistance to colchicine. An antibody directed against a synthetic oligopeptide corresponding to the amino-acid sequence (Glu-393-Lys-408) of P-glycoprotein from human mdr1 cDNA was prepared in rabbits. With immunoblotting and immunoprecipitation, we detected a 140-170 kDa protein in KB-C2 cells but not in parental sensitive KB cells. KB-C2 cells made a 125 kDa precursor that was slowly processed (t1/2 = 45 min) to the mature form of 140-150 kDa. The processing rate of P-glycoprotein was slower than that of low-density lipoprotein receptor. We detected another 160-180 kDa smear band, which might be a completely denatured form of P-glycoprotein. With immunoblotting, a minor band of high molecular mass (greater than 500 kDa) was also detected and this form increased after the cells were treated with chemical cross-linker, 1,5-difluoro-2,4-dinitrobenzene. The half-life of P-glycoprotein was long; no significant loss of P-glycoprotein was observed within 24 h after synthesis. Cells treated with tunicamycin produced a 120 kDa form of P-glycoprotein which was no longer processed but showed stability similar to that of the mature 140-150 kDa form. Agents that reverse multidrug resistance, phorbol ester and transport substrate did not affect the stability of P-glycoprotein.     

Purified protein kinase C phosphorylates microtubule-associated protein 2

We have investigated actions of purified protein kinase C on microtubule- and microfilament-related proteins. Among the cytoskeletal proteins examined, microtubule-associated protein 2 (MAP2) was found to serve as a good substrate. Other cytoskeletal proteins, tubulin, fodrin, cofilin, tropomyosin, and 53,000-Da protein, were very poorly phosphorylated. The amino acid residues of MAP2 that were phosphorylated by the protein kinase C were almost exclusively serine. The peptide mapping analysis indicated that protein kinase C and cAMP-dependent protein kinase phosphorylate MAP2 differently. The ability of MAP2 to interact with actin was markedly reduced by this protein kinase C-mediated phosphorylation. These data raise the possibility that phosphorylation of MAP2 by activated protein kinase C may be involved in cell-surface signal transduction.     

Interaction of digitonin and its analogs with membrane cholesterol

The interaction of digitonin with membrane cholesterol was studied by using various digitonin analogs, and radioactive desglucodigitonin. The following results were obtained concerning the effect of digitonin on erythrocytes, granulocytes and liposomes. Digitonin and its analogs showed activity to induce hemolysis, granulocyte activation and liposomal membrane damage. The activity was affected by change of the carbohydrate residue of the molecule; the order of hemolytic activity was digitonin greater than or equal to desglucodigitonin much greater than glucosyl-galactosyl-digitogenin greater than galactosyl-digitogenin, digitogenin. The relative activities of these compounds to induce granulocyte activation and liposomal membrane damage were similar to those observed in the hemolysis. [3H]Desglucodigitonin could bind to cholesterol in liposomes. The binding was stoichiometric and the ratio of desglucodigitonin bound to liposomes/cholesterol in liposomes was close to 1, irrespective of the cholesterol content in liposome. Damage to liposomes was, however, induced by desglucodigitonin only when they contained more than 0.2 molar ratio of cholesterol to phospholipid. Addition of digitonin as well as desglucodigitonin to preformed liposomes deprived of cholesterol affected the anisotropic molecular motion of spin-labeled phosphatidylcholine incorporated into the liposomes, suggesting that the molecules could be inserted into the lipid bilayer free of cholesterol. Molecules of desglucodigitonin in the lipid phase may, however, be equilibrated with those in the aqueous phase, unless they form a complex with cholesterol, since no appreciable amount of [3H]desglucodigitonin could be detected in the liposome fraction after separation by column chromatography. Digitonin decreased the order parameter of spin-labeled phosphatidylcholine when liposomes contained equimolar cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pacemaker Potentials for the Periodic Burst Discharge in the Heart Ganglion of a Stomatopod, Squilla oratoria

From somata of the pacemaker neurons in the Squilla heart ganglion, pacemaker potentials for the spontaneous periodic burst discharge are recorded with intracellular electrodes. The electrical activity is composed of slow potentials and superimposed spikes, and is divided into four types, which are: (a) "mammalian heart" type, (b) "slow generator" type, (c) "slow grower" type, and (d) "slow deficient" type. Since axons which are far from the somata do not produce slow potentials, the soma and dendrites must be where the slow potentials are generated. Hyperpolarization impedes generation of the slow potential, showing that it is an electrically excitable response. Membrane impedance increases on depolarization. Brief hyperpolarizing current can abolish the plateau but brief tetanic inhibitory fiber stimulation is more effective for the abolition. A single stimulus to the axon evokes the slow potential when the stimulus is applied some time after a previous burst. Repetitive stimuli to the axon are more effective in eliciting the slow potential, but the depolarization is not maintained on continuous stimulation. Synchronization of the slow potential among neurons is achieved by: (a) the electrotonic connections, with periodic change in resistance of the soma membrane, (b) active spread of the slow potential, and (c) synchronization through spikes.     

Peroxyoxalate chemiluminescent assay for oxidase activities based on detecting enzymatically formed hydrogen peroxide

A sensitive peroxyoxalate chemiluminescent (PO-CL) assay for activities of oxidases (uricase, choline oxidase, cholesterol oxidase and xanthine oxidase) which catalyse a formation of hydrogen peroxide was developed using 4,4′-oxalyl-bis[(trifluoromethylsulphonyl)imino]trimethylene-bis(4-methylmorpholinium)trifluoromethanesulphonate as a chemiluminogenic reagent and 2,4,6,8-tetramorpholinopyrimido[5,4-d]pyrimidine as a fluorophore. The standard curve for hydrogen peroxide was linear over the range 1 × 10^?7-1 × 10^?4 mol/L. Relative standard deviations for oxidase assays were 5.1–12.7% (n = 10). Detection limits were 1 × 10^?3 U/mL for uricase, 5 × 10^?4 U/mL for choline oxidase, 5 × 10^?3 U/mL for cholesterol oxidase and 5 × 10^?4 U/mL xanthine oxidase (sample to blank ratio, 3).     

15-Acetoxycostunolide from Magnolia sieboldii

A new germacranolide isolated from M. sieboldii was shown to be 15-acetoxycostunolide by spectroscopic and chemical methods. ¹H NMR spin decoupling and NOE experiments in the presence of a lanthanide shift reagent were used for structure elucidation.